Dendrobium is one of the largest genera in the Orchidaceae, and D. officinale is used in traditional medicine, particularly in China. D. officinale seeds are minute and contain limited energy reserves, and colonization by a compatible fungus is essential for germination under natural conditions. When the orchid mycorrhizal fungi (OMF) initiates symbiotic interactions with germination-driven orchid seeds, phytohormones from the orchid or the fungus play key roles, but the details of the possible biochemical pathways are still poorly understood. In the present study, we established a symbiotic system between D. officinale and Tulasnella sp. for seed germination. RNA-Seq was used to construct libraries of symbiotic-germinated seeds (DoTc), asymbiotic-germinated seeds (Do), and free-living OMF (Tc) to investigate the expression profiles of biosynthesis and metabolism pathway genes for three classes of endogenous hormones: JA (jasmonic acid), ABA (abscisic acid) and SLs (strigolactones), in D. officinale seeds and OMF under symbiotic and asymbiotic conditions. Low concentrations of endogenous JA, ABA, or SLs were detected in the D. officinale-Tulasnella symbiont compared with the asymbiotic tissues. Gene annotation results suggest that the expression of DEGs  related to JA and ABA biosynthesis from D. officinale were down-regulated, while most of the key DEGs related to SL biosynthesis from D. officinale were up-regulated in the symbiotic germinated seeds compared with the asymbiotic germinated seeds. Moreover, in the OMF, we found a significantly up-regulated differential expression of the JA and ABA biosynthesis-related genes in the symbiotic interaction, with the opposite expression trends to those found in Dendrobium. This indicates that Dendrobium seed symbiotic germination may be stimulated by the apparent involvement of the OMF in the production of hormones, and relatively low concentrations of endogenous JA, ABA, or SLs might be maintained to promote the growth of the D. officinale-Tulasnella symbiotic protocorm-like body. These results will increase our understanding of the possible roles played by endogenous hormones in the regulation of the orchid-fungus symbiosis. Dendrobium species in their natural habitats [Bletilla striata associated with Tulasnella sp. at an early developmental stage, and the essential genes required for the establishment of a mutualistic relationship with AM fungi and/or rhizobia in most terrestrial plants were identified from B. striata [In the majority of orchid species, seed germination is largely dependent on mycorrhizal fungi under natural conditions because of the lack of nutritional reserves . Symbiothabitats . The prohabitats . Althoughabitats ,4. Many habitats . Althoughabitats ,6,7. Miu striata . These sCymbidum goeringii and a Rhizoctonia-like mycorrhizal fungi causes the release of hormones, which can promote the growth of C. goeringii seedlings [D. officinale [Oncidium sphacelatum at the green protocorm stage of seed development with Ceratobasidium sp., and this could possibly result in an enhanced JA biosynthesis in the green protocorms with the fungal symbiont [Dendrobium.When mycorrhizal fungi undergo symbiotic interactions with orchids, phytohormones produced by the orchid and/or the microbial partners could play key roles ,10,11. Teedlings . The resficinale . The rolsymbiont . These sd-xylulose-5-phosphate (DXP) pathway is converted to geranylgeranyl pyrophosphate (GGPP), from which carotenoids are synthesized [Botrytis cinerea, Cercospora rosicolla, and some other species of fungi [trans-diol-ABA is the main precursor of ABA in B. cinerea and that ∆BcABA4 transformants accumulate 1′,4′-trans-diol-ABA [BcABA4 gene encodes a putative SDR (short-chain-type dehydrogenase/reductase) which is involved in the ABA biosynthesis in Arabidopsis [In higher plants, ABA is derived from the oxidative cleavage of carotenoids. Isopentenyl diphosphate (IPP) formed in the 1-deoxy-thesized ,14. The thesized . Fungi sthesized ,17. Afteof fungi ,19,20. Pbidopsis .S)-hydroperoxy linoleic or α-linolenic acid. This is then followed by the first committed step of JA biosynthesis catalyzed by allene oxide synthase (AOS) that converts 13-HPOT to a highly reactive allene oxide, which is converted to cis-(+)-12-oxophytodienoic acid by allene oxide cyclase (AOC). cis-(+)-OPDA is then transported to the peroxisome, where 12-oxophytodienoate reductase 3 (OPR3) and three rounds of β-oxidation convert it to JA [The JA biosynthesis pathways of plants and OMF are more similar. JA synthesis is initiated in the plastid by the oxygenation of linoleic or α-linolenic acid by lipoxygenases (LOXs) to form  species which are released from the roots and induce germination in seeds of the the soil ,23. SLs the soil , which wthe soil ,23,24,25the soil ,23. A hyynthesis . D14/DADD. officinale seeds in an effort to gain possible functional insights into the biosynthesis and metabolism of ABA, JA, and SLs in the regulatory module in the Dendrobium–Tulasnella symbiosis. We also predicted some unigenes involved in phytohormone production by the symbiotic fungus Tulasnella sp., and these genes had different expression profiles in symbiotic and asymbiotic germinated seeds.These phytohormones are well known to control plant development and to trigger important signaling events . MoreoveD. officinale germinated seeds and Tulasnella sp., while in the group of seeds germinated without fungi with a low germination rate (0.1%), we did not find hyphae, and these were therefore defined as asymbiotic germinated seeds (Do).Both morphological observation and Trypan blue staining, which showed that the intracellular fungal hyphae stained blue in the symbiotic germinated seeds DoTc; , indicatD. officinale and its Tulasnella endosymbiont with enhanced sequence coverage, nine cDNA libraries were constructed using RNA extracted from DoTc, Do, and free-living Tulasnella mycelium (Tc). In total, 667,270,886 Illumina paired-end raw reads were generated. The raw reads are available in the NCBI SRA database under the accession number SRP136098. After discarding the adaptor and low-quality sequences, we obtained 642,119,290 clean reads (accounting for 96 Gb of DNA sequence). The percentages of clean reads among the raw tags (Q20) ranged from 95.52% to 96.11%  and symbiotic and asymbiotic mycorrhizal fungi (DoTc vs. Tc). A total of 3367 DEGs  were obtained that included 2230 up-regulated and 1137 down-regulated genes in the DoTc vs. Do comparison , and 772D. officinale that were obviously up-regulated  samples, based on expression profiles determined from transcriptome sequencing. In the symbiotic roots of D. officinale seedlings (SR), these genes were all down-regulated compared to the control asymbiotic root samples (ASR), which showed expression trends that were opposite to the SGS samples, with the exception of CCD8-2. In the ABA and JA biosynthesis pathways, the expression of the ZXE, SDR-1 and SDR-2, LOX, AOS-1 and AOS-2, and OPR genes from D. officinale was significantly down-regulated in the SGS and SR samples compared to one or both of the asymbiotic control samples (Do and ASR). Moreover, we also assayed the differential expression of four BcABA genes  which could be involved in ABA biosynthesis in the OMF Tulasnella sp. in the SGS and SR samples and free-living Tulasnella mycelium (Tc). We found that the expression in the SGS and SR samples was significantly up-regulated compared with that in the Tc samples, and the expression of these four genes in the SGS samples was also based on the expression trends from the transcriptome sequencing and was more significantly upregulated than in the SR samples.In the symbiotic and asymbiotic germinated seed samples, we successfully detected the expression of key genes related to ABA, JA, and SL biosynthesis, and the results are shown in Tulasnella mycelium, corresponding to the RNA-Seq libraries DoTc, Do, and Tc, respectively. The results showed that in the symbiotic D. officinale germinated seeds, the JA concentration was slightly higher than in free-living Tulasnella mycelium and much lower than in the asymbiotic germinated seeds  in the symbiotic germinated seeds, asymbiotic germinated seeds, and free-living ed seeds , and theed seeds . 5-DS, aed seeds .Oncidium sphacelatum in association with the fungal symbiont Ceratobasidium sp. [Tulasnella sp.  [Theobroma cacao L.) [Brassica rapa L.) [Epipactis helleborine [Calanthe tricarinata [D. officinale seeds, the ABA concentration was lower than in the control, indicating that low endogenous ABA levels may have positive effects on D. officinale symbiotic seed germination. Previous studies have shown that in symbiotic D. officinale seedlings, the ABA concentration was lower than in the control [SDR and ZXE, two genes in the ABA biosynthesis pathway, was significantly suppressed in the symbiotic germinated seeds. These results agree with those of a recent study in which high concentrations of exogenously applied ABA were also found to suppress seed germination in vitro [Many studies have shown a negative correlation between the concentrations of endogenous ABA and rates of seed germination, which supports the notion that high concentrations of endogenous ABA in seeds may play a critical role in preventing germination in species such as wheat (ivum L.) , cacao (acao L.) , and raprapa L.) . In orchleborine , and Calcarinata . Our res control , which iin vitro .Tulasnella was significantly up-regulated, as was the transcription of some critical ABA biosynthesis unigenes with homologs in Botrytis; examples are two putative BcABA4 unigenes (Cluster-2641.12008 with 7.24-fold up-regulation and Cluster-2152.0 with 2.42-fold up-regulation) and two putative P450 monooxygenase protein CYP51 genes  in the symbiotic germinated seeds compared to the asymbiotic fungus Tulasnella sp.  and five ABA receptor PYL-related unigenes (6.54- to 10.06-fold up-regulation) were also found to be significantly up-regulated in the symbiotic germinated seeds compared with the asymbiotic Tulasnella sp. fungus. There is also emerging evidence to suggest that ABA plays an important role in compatible interactions in mutualistic plant-microbe interactions [Tulasnella sp. Based on these results, we can hypothesize that the symbiotic fungus Tulasnella produces more activated ABA to promote the recognition and establishment of compatible interactions with its mutualist orchid partner in early seed germination (stages 1–3); it is during these early stages that an abundance of pelotons is usually formed. After protocorm formation, the germinated seed develops to the fourth stage (emergence of leaf from shoot region), the reinvaded hyphae conspicuously decrease, and the hyphae are degraded in the symbiotic orchid cell [CYP707A (Cluster-3911.0), from Tulasnella that is highly expressed in symbiotic germinated seeds but not in asymbiotic Tulasnella, and this enzyme can degrade and deplete endogenous ABA levels to regulate intracellular ABA homeostasis in plants [D. officinale seed germination and inhibit the growth of Tulasnella mycelia.In our study, the relative transcription of most of the key MVA pathway genes from ella sp. , indicatm growth . In Cerainvasion . Moreoveractions , and it ractions . Howeverhid cell . Thus, tn plants ,35. In ln plants . At thisD. officinale, and in addition, we found that genes encoding key enzymes involved in the conversion of β-carotene to carlactone and mature SLs, D27, CCD7, CCD8, and MAX1 are expressed in both types of germination seeds, indicating that SLs might also play important roles in regulating D. officinale seed germination. Moreover, the content of 5-DS in the asymbiotic germinated seeds (2.03 ng/g·FW) was much higher than in the symbiotic germinated seeds (0.81 ng/g·FW), and this result was supported by the slow growth rate of the asymbiotic germinated seeds compared with that of the symbiotic germinated seeds, indicating that high concentrations of SLs or their biosynthetic precursors might inhibit growth. In other plants, studies have shown that SLs or their biosynthetic precursors act as plant hormones that inhibit shoot branch growth [Tulasnella fungus within orchid tissues to maximize the number of pelotons formed. This model might be supported by previous studies showing that SLs also promote branched growth in AM fungi [Tulasnella might suppress the biosynthesis of orchid SLs in order to maintain a low concentration of SLs to accelerate the germination rate in the D. officinale-Tulasnella symbiosis. Additionally, the germinated seed developed to the fourth stage (emergence of the leaf from the shoot region), the reinvaded hyphae conspicuously decreased, and the hyphae degraded in the symbiotic orchid cell [Tulasnella mycelium.In our study, we also detected a precursor of SLs (5-DS) in the germinated seeds of h growth ,23,24,25AM fungi ,23. Nevehid cell . At thisArabidopsis [Previous studies have suggested that ABA is one of the regulators of the strigolactone biosynthesis that acts through an as-yet-unknown mechanism . Moreovebidopsis ,38. ThisD. officinale plants were grown in the greenhouse of the Chinese Academy of Forestry  at day/night temperatures of 24 °C/18 °C, a relative air humidity of 65–75%, a 10-h day/14-h night photoperiod, and an irradiance of 700 mol m−2·s−1. Six D. officinale seed capsules were collected from the greenhouse and divided into two groups after surface sterilization. Seeds from the first group were cultured with Tulasnella sp., which was isolated from Cymbidium goeringii roots ; a BLAST search showed that the ITS sequences from this isolate were 99% homologous to the ITS from T. calospora isolate Pv-PC-2-1 (NCBI accession No: GU166418.1) [Tulasnella sp. were cultured on 8 g/L oatmeal agar (OMA) medium; this group was defined as DoTc. The second group of seeds was cultured on the same OMA medium without fungi. Seeds were sown and incubated according to the procedures described by Xu et al. [D. officinale and Tulasnella sp. The group of seeds germinated without fungi had a very low germination rate (0.1%), and we did not find fungal hyphae; these were defined as asymbiotic germinated seeds (Do). Free-living fungal mycelium was maintained on an OMA medium at 25 °C for 10 days (Tc). DoTc, Do, and Tc  were collected, immediately frozen in liquid nitrogen, and stored at −80 °C prior to RNA extraction.66418.1) . The seeu et al. . The DoTu et al. ,34, at wu et al. ,40. Micr® spectrophotometer . RNA concentration was measured with the Qubit® RNA Assay Kit in the Qubit® 2.0 Fluorometer . RNA integrity was assessed using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system .The total RNA was extracted from each sample using the RNeasy Plant Mini Kit  and treated with an RNase-free DNase I digestion kit  to remove residual genomic DNA. RNA degradation and contamination was monitored by electrophoresis on 1% agarose gels. RNA purity was checked using the NanoPhotometer® Ultra™ RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations, and index codes were added to attribute the sequencing reads to each sample. The PCR products were purified (AMPure XP system) and the library quality was assessed on the Agilent Bioanalyzer 2100. The RNA-Seq libraries were sequenced on an Illumina Hiseq 4000 instrument to generate 150 bp paired-end reads. All samples were sequenced with three biological replicates.For each sample, 3 µg of the total RNA was used as input material for the RNA-Seq library preparations. Sequencing libraries were generated using the NEBNextN-containing reads, and low quality reads from the raw data. Additionally, the Q20 and Q30 scores, and the GC content of the clean reads were calculated simultaneously. All downstream analyses were based on high-quality clean data.Raw reads in the FASTQ format were first processed using in-house custom Perl scripts. In this step, clean reads were obtained by removing reads containing adapters, poly-D. officinale Reference Genome: The index of the D. officinale reference genome [Sequence read mapping to the e genome  was buile genome  with thee genome . All assD. officinale gene. The FPKM (expected number of Fragments Per Kilobase of transcript sequence per Million base pairs sequenced) was then calculated for each gene based on the length of the gene and the read count mapped to the gene [HTSeq v0.6.1 was used to count the read numbers mapped to each the gene .Gene expression levels in the OMF were estimated using RSEM with the default settings . The clep-values were adjusted using the Benjamini–Hochberg procedure for controlling the false discovery rate. Genes with a log2(Fold_Change) > 1 and adjusted p-value < 0.001 identified by DESeq were considered to be differentially expressed.A differential expression analysis of the two conditions/groups  was performed using the DESeq R package (1.18.0) with the default settings . DESeq phttp://www.genome.jp/kegg/). We used the KOBAS [Unigene function was annotated based on the seven databases reported by Liu et al. . Metabolhe KOBAS  softwareThe concentrations of the ABA, JA, and SL hormones in the Do, DoTc, and Tc samples were measured using ultraperformance liquid chromatography coupled with a tandem quadrupole mass spectrometer equipped with an electrospray interface (UPLC-ESI-qMS/MS). Six biological replicates were used in the hormone quantification measurements.2. The crude extracts were dissolved in 3 mL normal hexane and filtered through spin columns  with 5 mL of normal hexane, and the elution products were then evaporated to dryness in a stream of N2. The crude extracts were then dissolved in 200 μL of methanol and filtered through spin columns , and subjected to an HPLC-MS/MS analysis.1. 5-DS: To isolate 5-DS, 1 g (fresh weight) samples of symbiotic or asymbiotic seeds were frozen in liquid nitrogen, ground to a powder, transferred to 2 mL microcentrifuge tubes, and then extracted with 10 mL acetone with ultrasound in a water bath for 15 min, after which the tubes were kept at −20 °C for at least 12 h. After centrifugation at 8000 rpm for 5 min, the supernatants were transferred into new tubes and centrifuged at 13,000 rpm at 4 °C for 5 min. The lower organic phases were then transferred to new tubes and evaporated to dryness in a stream of dry Nm/z 331.0/234.1 with a declustering potential (DP) of 45 V and collision energy (CE) of 14 V.Identification of 5-DS by LC–MS/MS: HPLC separation was conducted with an Agilent 1290 HPLC instrument (Agilent Technologies Corporation) fitted with a poroshell 120 SB-C18 column , and 2 µL was injected for the HPLC-MS/MS analysis. The column temperature was kept at 30 °C. The binary mobile phase system consisted of methanol (A) and water (B) in 0.1% formic acid under a gradient condition  with a flow rate of 0.3 mL/min. Mass spectrometry was performed with a SCIEX-6500Qtrap (MS/MS) mass spectrometer  equipped with an electrospray source. The drying and nebulizing gas was nitrogen generated from pressurized air in an N2G nitrogen generator . The curtain gas was set to 25 psi, the nebulizer gas was set to 55 psi, the heater gas was set to 65 psi, and the ion spray voltage was set to 5000 V. The atomization temperature was set to 350 °C. Analytes were quantified in the multiple-reaction-monitoring (MRM) mode for the known 5-DS detection, at 2. The extracts were dissolved in 400 μL of methanol and filtered through spin columns , and subjected to an HPLC-MS/MS analysis.2. ABA and JA: To isolate the hormones, 1 g (fresh weight) samples of symbiotic or asymbiotic seeds were frozen in liquid nitrogen, ground to a powder, and transferred to 2 mL microcentrifuge tubes. The ground tissue was extracted in 10 mL isopropanol/hydrochloric acid buffer by shaking at 4 °C for 30 min, and 20 mL of dichloromethane was then added and the mixture was vibrated at 4 °C for a further 30 min. The tubes were centrifuged at 13,000 rpm at 4 °C for 5 min, and the lower organic phase was transferred to a new tube and was then evaporated to dryness in a stream of Nv/v) methanol, 0.05% formic acid, and 5 mmol/L ammonium acetate in water (A) and acetonitrile (B) under a gradient condition , 4–5 min, 100% (B)) with a flow rate of 0.3 mL/min. The sample chamber temperature was set to 10 °C and the injection volume was 10 μL. The capillary voltage was controlled at 20 V. The ion source temperature was set to 150 °C, the cone gas flow was 50 L/h, desolvation temperature was 400 °C, and the desolvation gas flow was 800 L/h. Analytes were also quantified in multiple-reaction-monitoring (MRM) mode for the known ABA and JA detections.ABA and JA were measured with an HPLC-ESI-qMS/MS instrument  fitted with an AQUITY UPLC BEH C18 column . The column temperature was kept at 40 °C. A binary mobile phase system consisting of 2%  that were previously used to prepare the RNA-Seq libraries were used to perform qRT-PCR assays for gene expression quantification. The plant materials were flash-frozen in liquid nitrogen and stored at −80 °C prior to RNA extraction. RNA isolation, qRT-PCR, and calculations of relative mRNA levels were performed as previously described [DoEF1α and DoRPL30 [D. officinale, and TcEF1α [TcTUBB (Cluster-2641.19171, Tulasnella were used as reference genes for the normalization of gene expression, respectively. The primer sequences are shown in The symbiotic and asymbiotic root samples from the escribed . Each da DoRPL30  from D. d TcEF1α  and TcTUD. officinale germinated seeds to examine the expression profiles of genes related to the biosynthesis of three types of endogenous hormones—JA, ABA, and SLs—in the D. officinale-Tulasnella symbionts, asymbiotic germinated seeds, and the free-living OMF. We also performed comprehensive analyses of the contents of individual hormones, and assayed gene expression using qRT-PCR in the symbiotic and asymbiotic seed samples. The results allowed us to propose potential regulatory mechanisms for JA, ABA, and SLs from the orchid and OMF in the D. officinale-Tulasnella symbionts. Our study will provide new insights into the molecular mechanisms that regulate the orchid-fungus symbiosis.In this study, we generated transcriptome datasets of


Background: Dialysis is the first procedure to partially replace renal function in end-stage renal diseases, despite several adverse side effects, such as infections. The primary aim of this study was to evaluate the levels of immune CMV-specific CD8+ T cells in a representative cohort of pre-transplant patients receiving hemodialysis (HD) or peritoneal dialysis (PD). The secondary aim was to monitor the CMV-specific CD8+ T cells in kidney transplant recipients undergoing different types of dialysis during the first year following their transplant.Methods: Sixty-nine patients were enrolled and examined with respect to the type of dialysis they received. HLA class I dextramers for CMV were used to determine the quantity of CMV-specific CD8+ T cells. The CMV DNA viral load was also determined. Forty-two of the patients enrolled in the study underwent solid organ transplantation and were analyzed during their first year post-transplantation.Results: Patients receiving HD had fewer CMV-specific CD8+ T cells than those in PD (p < 0.05). We also observed that patients in PD had more CMV-specific CD8+ T cells during the follow-up period than those in HD (p < 0.05), independently of the CMV DNA. Finally, PD patients had a higher frequency of CD8+ Effector-Memory RA T cells (TEMRA) and a lower frequency of central memory T cells (TCM) than did HD patients.Conclusions: These results indicate the better status of CMV-specific T cell immunity in PD patients. The use of CMV T cell dextramers would be advantageous for monitoring the CD8+ T-specific response, enabling the use of prophylactic treatment to be optimized. Patients in end-stage renal diseases experience disturbances of the immune system and are highly susceptible to infections arising from dialysis. The frequency of mortality of patients in dialysis is higher than in the general population, especially in patients in hemodialysis (HD) compared with those in peritoneal dialysis (PD) , 2. The Two main strategies are used to prevent CMV infection after kidney organ transplantation . The firCustomarily, CMV-DNA copy number has been used to identify viral reactivation , but theThe aim of this study was to analyze the impact of the type of dialysis on the frequency of CMV-specific CD8+ T cells in pre- and post-transplant patients. Our overall purpose was to redefine therapeutic strategies for selected groups of immunosuppressed patients.A cohort of 69 unrelated Caucasian patients on the waiting list for a kidney transplant in the Nephrology Service of the Hospital Universitario Central de Asturias (HUCA), Oviedo, Spain, were included in the study between February 2016 and February 2017 . They weBlood samples were extracted in a blood collection tube with EDTA at different times .This study was carried out in accordance with the recommendations of the Regional Ethics Committee of Clinical Research of the Principado de Asturias. All the subjects gave their written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of our hospital.CMV viral serostatus of the patients was determined as part of current routine clinical practice in the Microbiology Service (HUCA) using the Liaison CMV IgG II assays . In-house real-time PCR was used to quantify CMV DNA in accordance with general regulatory guidelines concerning CMV viral load monitoring. A blood sample was extracted in a blood collection tube with EDTA, and the viral DNA was isolated from peripheral blood mononuclear cells (PBMCs). The viral β-glycoprotein and the human APOB gene as a control were amplified and hybridized with fluorescent-labeled probes in an RT-PCR based on TaqMan Genotyping Assays . The viral load in copies of CMV DNA per mL was determined, from which the International Units (IU) per mL were then calculated. The CMV DNA data of the patients were collected at the same post-transplant times as for the CMV-specific CD8+ T cell determination.TM Multi-Check control, BD Biosciences, San Jose, CA, USA). Absolute CD8+ T cells number and percentage were calculated using Trucount Tubes .The Dextramer CMV kit  was used according to the manufacturer's instructions to quantify the CMV-specific CD8+ T cells. The CMV-MHC class I dextramers used, which covered most HLA specificities, were A:0101, A:0201, A:0301, A:2402, B:0702, B:0801, and B:3501. Samples were acquired using a Gallios flow cytometer  and analyzed using Kaluza Analysis software . A whole-blood control for lymphocyte subset enumeration was used in all sessions , central memory (CM) (CCR7+CD45RA–), effector memory (EM) (CCR7–CD45RA−), and effector memory RA (EMRA) (CCR7–CD45RA+) CD8+ T cells were assessed by flow cytometry using the following antibodies: CD3 , CD8 , CD45RA , and CCR7 .−3M l-glutamine and Hepes, supplemented with 10% FCS  and antibiotics. For the ELISpot assay, 3 ×105 PBMCs per well were placed in triplicate wells of a 96-well filter plate  that was coated with anti-IFN-γ Ab . These cells were stimulated with CMV-pp65  and with 1 μg/mL anti-PMA  as a positive control, and incubated for 24 h at 37°C. Negative controls were also included using PBMCs plus medium and DMSO. Plates were incubated for 2h at room temperature with 100 μL (1 μL/mL) biotinylated detection IFN-γ antibody to detect IFN-γ captured by the plate-bound Ab. Subsequently, plates were incubated with streptavidin (1 μg/mL) for 2 h at room temperature. As a final step, spots were developed by adding 200 μL of 3-amino-9-ethylcarbazole  in acetate buffer supplemented with H2O2 30% for 3–5 min. Resulting spots were counted using a computer-assisted ELISpot reader . Positive ELISpot signals were defined as those containing at least 25 spot-forming units per well.CMV-specific T cell activity was determined by measuring IFN-γ upon stimulation of PBMCs in 43 patients. The PBMCs were isolated from 5 mL of citrate blood using a standard Ficoll–Paque density gradient and resuspended in RPMI 1640 medium containing 2 ×10A descriptive statistical analysis of the variables was done separately for pre-transplant and post-transplant patients who received HD or PD. The continuous numerical variables were not normally distributed, so they were summarized as their median and range. Absolute and relative frequencies are displayed for qualitative variables. In order to test the difference between dialysis groups, the non-parametric Wilcoxon rank-sum test was used for continuous measures, while Fisher's exact test was considered for qualitative variables.p < 0.05 in all analyses. Data were analyzed within the R statistical environment (version 3.6.0).Multiple linear and logistic regression models were fitted as a multivariate study of the simultaneous statistical associations among several of the variables studied. Model terms were chosen in a backwards-stepwise manner, considering the Akaike information criterion (AIC), and the parameter estimates with 95% confidence intervals and associated probabilities were reported for the final model. The distribution of those measurements showing statistically significant differences between groups was displayed using boxplots. Statistical significance was concluded for values of n = 69; p = 0.03; p = 0.012; Patients were classified according to the type of dialysis they were receiving n = 69; : 59.4% op < 0.01) and serological CMV status (p < 0.005) were significantly related to the frequency of anti-CMV CD8+ T cells  as regressors in the initial model. The final stepwise regression model shows that the type of dialysis  compared with those in PD, 85% of whom  showed preformed CMV-specific T cells directed to CMV-pp65. These differences were not significant.In relation to the CD8+ T cells subsets, we found that patients in PD tended to have a higher porcentage of CD8+ TEMRA cells  and a lower frequency of TCM cells than patients in HD , but the magnitude of the difference was not sufficiently great to be statistically significant in the small sample of patients analyzed. Finally, in order to determine the immune responsiveness of these specific T cells we analyzed the levels of pre-transplant IFN-γ using a CMV ELISpot assay. Patients in HD exhibited preformed CMV-specific T cells directed to CMV-pp65 in 73.9% of the cases analyzed  for 1 year. Of the 42 patients, 19 experienced CMV reactivation (>100 IU/mL) within 360 days post-transplantation —a similar finding to that noted in pre-transplanted patients. Furthermore, during the follow-up period, patients who were receiving PD had significantly more CMV-specific CD8+ T cells than did those in HD . The differences were greatest after 90 days, but frequencies had equalized by 360 days. Considering the patients with respect to the CMV reactivation, the CMV-specific CD8+ T cells frequency at the times studied showed that values for PD patients were more variable, but generally higher, than those patients in HD in both patient groups  of the patients who experienced CMV reactivation and needed antiviral treatment were from the group who had received HD, compared with 26.2% (5 of 19) of the patients who had received PD. There were fewer than 5 CMV-specific CD8+ T cells/μl in this group at the beginning of the CMV reactivation. In contrast, 5 of 19 patients who underwent CMV reactivation did not receive antiviral prophylaxis or treatment. These patients (2 HD and 3 PD patients) had a high frequency of >20 CMV-specific CD8+ T cells/μl during follow-up.Finally, other clinical parameters related to CMV reactivation, such as the CMV serostatus of the donors and recipients, the immunosuppressor levels and receipt of induction therapy were analyzed, but did not reveal any differences in the frequency of CMV-specific CD8+ T cells. A multivariate analysis including the type of dialysis, age, time on dialysis, serological CMV status, and total frequency of CD8+ T cells was performed, but none of the variables proved to be significantly associated with the increase in frequency of anti-CMV CD8+ T cells, possibly due to the low number of patients analyzed.In summary, the frequency of CMV-specific CD8+ T cells increased in both groups of patients by 90 days, possibly due to the reduction in immunosuppression levels and the end of CMV prophylaxis. We also observed that patients in PD had better cellular immune status than those in HD.CMV has a negative consequence for patients and allograft outcomes after kidney transplantation. CMV-specific CD8+ T cells are thought to be crucial to the control of CMV replication in the early phase after kidney transplantation . The freThe aim of this study was to monitor the frequency of CMV CD8+ specific T cells by using the dextramer technology in pre- and post-kidney transplant patients. We found more CMV-specific CD8+ T cells in patients in PD than in those receiving HD, and noted that this status was maintained throughout the 1-year follow-up after kidney transplantation, irrespective of patients' CMV serological status. Thus, protective anti-CMV cellular immunity may exist in these patients, unlike those receiving HD, indicating that patients in PD treatment were better able to resolve CMV reactivation. During the post-transplant follow-up, the presence of CMV-specific CD8+ T cells was not sufficient to prevent viral reactivation (CMV viremia) in most patients. Associated risk factors and a greater susceptibility to immunosuppressors led to the decline in the frequency of CMV-specific CD8+ T cells. This is insufficient during the initial months to provide further protection from CMV replication, and valganciclovir prophylaxis is necessary, at least for the first 3 months, in patients with risk factors such as D+/R– transplants. On the other hand, some patients with a high frequency of CMV-specific CD8+ T cells, most of whom are patients in PD, are able to control the viremia themselves without the need for antiviral treatment. Nevertheless, this variation between patients suggests that CMV-specific T cell monitoring could provide a means of risk stratification in addition to CMV serostatus.The type of dialysis is known to have a range of effects on the immune response . Mild moCMV infection generally leads to substantial and long-lasting changes in circulating T cells. In particular, those of the highly differentiated TEMRA (Effector Memory RA) cells subset are expanded in the CD8+ T cell population . RecentlThe determination of cellular immunity to assess risk before kidney transplantation by analyzing patients on the waiting list who are receiving dialysis, and the analysis of immune responsiveness using ELISpot, are powerful methods for evaluating CMV antiviral prophylaxis therapy. Further studies are needed to identify the variables associated with the risk of CMV reactivation, to characterize these CMV-specific T cells phenotypically, and to establish a cut-off for pre-transplant CMV-specific CD8+ T cells in order to be able to categorize patients.The raw data supporting the conclusions of this manuscript will be made available on request to the corresponding author.This study was carried out in accordance with the recommendations of the Regional Ethics Committee of Clinical Research of Principado de Asturias, with written informed consent from all subjects. The study was carried out in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics committee of our hospital.JV-C, CD-C, and CL-L: concept and design. JV-C, VC-I, LS-D, and SM: experiments and procedures. JV-C, VC-I, CD-C, and CL-L: writing of article. SP-F: statistical analysis. LS-D, SM, and CD-C: patients selection.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In untreated children, lyso-Gb1 levels were inversely correlated with platelet counts. During follow-up, lyso-Gb1 increased in almost 50% of untreated children, more commonly in younger children. In treated children, lyso-Gb1 levels were inversely correlated with hemoglobin levels. The increase of lyso-Gb1 while receiving ERT, seen in eight children, was partly associated with compliance and weight gain. Lyso-Gb1 seems to be a useful biomarker for monitoring children with GD and should be included in the routine follow-up. Progressive increase in lyso-Gb1 levels in untreated children suggests ERT initiation.The role of glucosylsphingosine (lyso-Gb1), a downstream metabolic product of glucosylceramide, for monitoring treated and untreated children with Gaucher disease (GD) has not yet been studied. We reviewed the clinical charts of 81 children (<18 years), 35 with mild type 1 GD (GD1), 34 with severe GD1 and 12 with type 3 GD (GD3), followed at Shaare Zedek Medical Center between 2014–2018. Disease severity for GD1 was based on genotypes. Forty children (87%) with severe GD1 and GD3 received enzyme replacement therapy (ERT) compared to two children (6%) with mild GD1. Lyso-Gb1 measurements were conducted on dried blood spot samples taken at each clinic visit. Lyso-Gb1 levels were significantly lower in children with mild compared to severe GD1 ( Gaucher Disease (GD) is among the most prevalent, recessively inherited, lysosomal storage disorder caused by a deficiency in the enzyme β-glucocerebrosidase. The deficient enzymatic activity, results in the lysosomal accumulation of its substrate glucosylceramide, most prominently in macrophages . GlycospIn the past, the majority of GD cases manifested during childhood and adolescence, with almost half of the cases diagnosed before ten years of age . In earlGlucosylsphingosine (lyso-Gb1), a deacylated form of glucosylceramide, is also degraded by the glucocerebrosidase. Lyso-Gb1 was proved to be a highly sensitive and specific biomarker for diagnosis and monitoring of adults patients with GD ,9. This p = 0.009) and not different from children with GD3 (p = 0.81). No significant differences were found between children with mild and severe GD1 regarding the age, gender, platelet count, hemoglobin levels and spleen and liver MN  volume. Significantly larger spleen and liver MN volume were found in children with GD3 compared to those with GD1 .The clinical phenotype of all children at their last visit is presented in p < 0.0001, r = −0.42) and hemoglobin levels , but not with liver MN volume, spleen MN volume, child’s age, and weight. When analyzing according to genotype, a significant correlation between lyso-Gb1 and platelet was found only in children with mild GD1 and a significant correlation between lyso-Gb1 and hemoglobin was found only in children with severe GD1. Similarly, when analyzing according to treatment status, irrespective of genotype, a significant inverse correlation was found between lyso-Gb1 and platelet count only in untreated children (p = 0.002 vs. p = 0.178 [in treated children]) and between lyso-Gb1 and hemoglobin level only in treated children (p = 0.01 vs. p = 0.197 [in untreated children])  .p = 0.0003).Pre-ERT data, available for ten children, were compared to the first visit data of the 28 untreated children . As expep = 0.007)  . Lyso-Gb= 0.007) . Retrosp= 0.007) A.p = 0.01)  B.Time of follow-up and weight change were not associated with the pattern of change of lyso-Gb1 levels in treated and untreated children.In this study, we show the potential value of lyso-Gb1 in children with GD both for monitoring untreated and treated children. An association between GD severity and lyso-Gb1 levels was expressed in several ways. First, an association between genotype and the lyso-Gb1 level was shown; significantly higher levels of lyso-Gb1 in children with severe GD1 compared to children with mild GD1 . Second,Association between genotype and lyso-Gb1 levels are consistent with adult data showing that patients with less severe disease, i.e., homozygotes N370S, generally show more modest increases in plasma lyso-Gb1 . As expeIn treated children, the lyso-Gb1 increase in some of the cases was associated with compliance and significant weight gain (>15%). We believe that the increase in lyso-Gb1 was related to lower ERT dose/kg and not the weight change per se. The linkage between the dose of ERT and response was previously shown in patients with GD .Recently, we have shown in adults with GD1 that lyso-Gb1 is a reliable response biomarker preceding changes in other disease parameters . InitiatIn children on ERT, who achieved a normal platelet count, lyso-Gb1 levels correlated only with hemoglobin levels; a parameter with a slower response to treatment. Similar findings were reported in the splenectomy era; in splenectomized patients, anemia was more prominent than thrombocytopenia, whereas, in non-splenectomized patients, thrombocytopenia is typically more prominent than anemia .In a study which included treated patients, mainly adults, age had a significant inverse correlation with plasma lyso-Gb1 . The autInterestingly, age did play a role in the pattern of change in lyso-Gb1 levels over time in mildly effected untreated children. A younger age at first visit was associated with an increase in lyso-Gb1 levels. At an older age, the lyso-Gb1 levels may plateau or even mildly drop. Extended follow-up into adulthood is needed for those untreated children. The fact that lyso-Gb1 could drop without treatment suggests that only a consistent and significant increase in lyso-Gb1 levels should lead to treatment decisions in children presenting with asymptomatic/mildly symptomatic disease.The additional interesting aspect of our study is the similarity between GD1 and GD3 concerning lyso-Gb1 levels. This might reflect the fact that the GD3 patients in our center belong to the so-called Type 3b . OthersEighty-one consecutive children (<18 years) with GD who visited the Gaucher Unit at Shaare Zedek Medical Center from July 2014 to December 2018 were included in this study. Lyso-Gb1 measurement had been included in the routine clinical and laboratory assessment during all follow-up  visits.Disease severity for this study was based on genotypes for GD1, i.e., mild GD1 were all children the with N370S/N370S or N370S/R496H genotypes and severe GD1 were N370S compound heterozygotes . Type 3 n = 81), the second was an analysis of data at first visit for untreated patients and patients with pre-treatment data (n = 38), and the last analysis was of patients data with more than one visit (n = 68). The delta of every measurement between the last and the first visit was calculated. Log transformation of lyso-Gb1 was done to achieve normal distribution. Results are presented as median and range. Correlations between the lyso-Gb1 levels and continuous measurements were tested by non-parametric Spearman's correlation and Pearson's correlation, for non-normal and normally distributed data, respectively. Mann–Whitney non-parametric test was used to compare non-normally distributed data in independent samples. Multivariate analysis was used to clarify the impact of ERT vs. non-ERT and impact of genotypes (mild vs. severe GD1) on the calculated correlation between the log of lyso-Gb1 and the other measurements. IBM SPSS version 25 was used for analysis. Results were considered to be statistically significant when two-tailed p-values were ≤0.01.For data analysis, we defined in our study three cohorts. The first was an analysis of data at last visit for all patients (The correlations found between lyso-Gb1 levels and disease severity as well as the changes in levels with and without therapy support the importance of using this biomarker in monitoring children with GD, both untreated and treated, for the need to start treatment and to follow the response to therapy, respectively. At this point, we cannot recommend a specific cut-off for lyso-Gb1 levels as a sole indication for beginning ERT, in part because by the time lyso-Gb1 levels became available many children were already on ERT. We believe that within the coming years, with a more significant number of naïve patients, we will be able to define a specific value. In the interim, we strongly recommend including lyso-GB1 in the routine follow-up of all children with GD and a progressive increment in lyso-Gb1 should lead to consideration of ERT in untreated children or to a dose increase in treated children.


Lactobacillus and Bifidobacteria, can prevent and treat allergic diseases. Prebiotic oligosaccharides, a food/diet additive, can enhance health and performance via modulating gut microbes and immune responses. The purpose of this study was to examine whether fructo-oligosaccharides (FOS) could alleviate soybean-induced anaphylaxis by modulating gut microbes. Piglets (21 days of age) were sensitized with a diet containing 5% soybean and 30% peeled soybean meal. The treatment with 0.6% FOS started 1 day prior to sensitization and continued everyday thereafter. Blood was collected for measurements of immune indices. The DNA samples isolated from fresh intestinal contents of the middle jejunum (M-jejunum), posterior jejunum (P-jejunum), ileum, and cecum were used for gene sequencing based on 16S rRNA. Our results showed that there was an increase of glycinin-specific IgG, β-conglycinin-specific IgG, total serum IgG and IgE, and occurrence of diarrhea in piglets sensitized with soybean antigen. There was a decrease in interleukin 4 (IL-4) and IL-10 and an increase of interferon-γ (IFN-γ) in piglets with FOS treatment, compared with the piglets without FOS treatment. Improvement of intestinal microbes was indicated mostly by the increase of Lactobacillus and Bifidobacteria in M-jejunum and the decrease of Proteobacteria in P-jejunum and ileum. The correlation analysis indicated that FOS treatment decreased those closely related to the key species of gut microbes. These results suggest that FOS can alleviate soybean antigen-induced anaphylaxis, which is associated with increased Lactobacillus and Bifidobacteria in M-jejunum and declined Proteobacteria in P-jejunum and ileum of piglets.Soybean-induced anaphylaxis poses a severe threat to the health of humans and animals. Some commensal bacteria, such as  Soybean is a species of legume and an ideal source of protein for humans and animals . HoweverPrevious studies have shown that traditional processing techniques such as heat treatment, high-pressure puffing, and fermentation  can passLactobacillus and Bifidobacteria, play a major role in maturation and homeostasis of the gut-associated immune system  barrows were weaned at 21 days of age and had an average weight of 6.81 ± 0.58 kg. The piglets were divided randomly into three groups of five each and housed in a livestock farmhouse located in Jilin Agricultural University, Changchun.The ingredients and nutrients of the diet are shown in Table After 7 days of adaptation, the piglets were treated at 28 days of age. The piglets in allergy group and FOS group were sensitized for the first 10 days by feeding allergy diet and subsequently boosted by feeding allergy diet on days 16–18 and on days 31–32. The control diet was given on all the other days. The piglets had free access to food and water and were weighed at the start and the end of the trial (day 0 and day 32) to calculate the average daily weight gain.The blood samples were obtained from each piglet by vena cava puncture using 10 mL gel vacuum collective tubes on days 10, 25, and 32. These blood samples were taken at a 3 h interval after feeding the allergy diet, incubated for 30 min at 37°C, and then centrifuged for 20 min at 2,000 rpm/min. The supernatant was collected and stored at -80°C for measurements of immunoglobulin and cytokine levels.At day 32, three piglets from each group were chosen randomly and euthanized. The contents of four intestinal segments including middle of jejunum (M-jejunum), posterior of jejunum (P-jejunum), ileum, and cecum were collected. These samples were snap freezing in liquid nitrogen and stored at -80°C until use.Total serum IgG and IgE antibody levels were determined by using swine ELISA kits  according to the manufacturer’s instructions. Glycinin-specific IgG and β-conglycinin-specific IgG antibody levels were determined by using an indirect ELISA as previously described by Concentrations of interleukin 4 (IL-4), IL-6, IL-10, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in serum were determined using the swine enzyme-linked immunosorbent assay kit  according to the manufacturer’s instructions.The microbial genomic DNA was extracted from intestinal content samples using Fast DNA SPIN extraction kits  according to the manufacturer’s instructions. The quantity and quality of extracted DNAs were measured using a NanoDrop ND-1000 spectrophotometer  and agarose gel electrophoresis, respectively. In this experiment, the microbes’ V3–V4 hypervariable regions of 16S rRNA gene, with a length of approximately 500 bp, were used for sequencing. The primer sequences were 338F 5′-ACTCCTACGGGAGGCAGCA-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′. The polymerase chain reaction (PCR) condition was as follows: initial denaturation at 98°C for 2 min, followed by 25 cycles consisting of denaturation at 98°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s, with a final extension of 5 min at 72°C. The PCR amplicons were purified with Agencourt AMPure Beads  and quantified using the PicoGreen dsDNA Assay Kit . After that, the individual quantification steps were pooled in equal amounts paired-end 2 bp × 250 bp sequencing was performed using the Illumina MiSeq platform with MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology, Co., Ltd. .Illumina MiSeq sequences were analyzed by Quantitative Insights Into Microbial Ecology, v1.8.0 (QIIME)  for taxop < 0.05 was considered statistically significant. Canonical correspondence analysis (CCA) was performed using Canoco for Windows 4.5 between the first 20 predominant genera and the immune indices in each group. Correlation analysis between key communities and immune indices were determined based on Pearson’s rank correlation coefficient.The data of immune indices were presented as mean ± standard deviation and analyzed using one-way analysis of variance (one-way ANOVA), followed by LSD multiple comparison tests by SPSS software (IBM SPSS Statistics 20 for windows). The value of p < 0.05) compared with the control group. However, supplementation with FOS increased the daily weight gain reaching values similar to the control group. Furthermore, occurrence of diarrhea decreased in the FOS group (8.75%), compared with the allergy group (22.50%).The performance and occurrence of diarrhea are shown in Table p < 0.05). Glycinin-specific IgG and β-conglycinin-specific IgG were significantly higher in the allergy group compared with the control group (p < 0.05); however, no differences were observed between the FOS group and the control group.To explore the effects of FOS on soybean-induced allergy in sensitized pigs, the total serum IgG, IgE, glycinin-specific IgG, and β-conglycinin-specific IgG levels were determined Table . The totp < 0.05), while IL-4 and IL-10 levels were significantly lower in the control and FOS groups compared with the allergy group (p < 0.05). There were no differences for IL-6 and TNF-α among different groups (p > 0.05)  (Table p < 0.05). As shown in Table p < 0.05). The Shannon indices ranged from 6.777 to 8.193 in three groups, respectively. There were no statistically significant differences in diversity estimators between M-jejunum and P-jejunum (p > 0.05).A total of 1,282,521 sequences were obtained from the piglets gut microbes, with an average of 35,626 sequences per sample . The bacterial diversity of the intestinal content samples in different groups is presented in Table 5) Table . The M-jhe phylum level, Firmicutes was the most-dominant phylum among all intestinal segments, followed by Proteobacteria, Actinobacteria, Cyanobacteria, and Bacteroidetes  . The abundance of Bacteroidetes increased more than 7–10-fold at cecum than other segments.At ts Figure . For P-j5) Table . In contLactobacillus made it the most-dominant bacterium in all genus  . Compared to the allergy group, Lactobacillus showed a dynamic pattern in the FOS group with a 41.48% of increase at M-jejunum, an 8.97% of decrease at P-jejunum, a 33.16% of increase at ileum, and a 22.51% of decrease at cecum. Bifidobacteria is a minor community in intestinal microbes, which was enriched in the FOS group in M-jejunum. The abundance of Streptococcus was significantly higher in the allergy group at P-jejunum and ileum (p < 0.05). Moreover, Clostridiaceae was significantly higher in the allergy group at M-jejunum and P-jejunum compared to the control group (p < 0.05), whereas it was significantly decreased in the FOS group compared with the allergy group. In addition, Turicibacter, Peptostreptococcaceae also changed as shown in Table At the genus level, a total of 183 genera were identified from all samples. The abundance of s Figure  and was Canonical correspondence analysis reflects the relationship between microbes and environmental factors (immune indices). As shown in Figure Firmicutes was correlated positively to the level of β-conglycinin-specific IgG in M-jejunum and was correlated negatively to the level of IL-10 in P-jejunum and glycinin-specific IgG in ileum (p < 0.05). Proteobacteria was correlated positively to the expression of IL-4 in P-jejunum and cecum and the level of IL-10 in P-jejunum, ileum, and cecum (p < 0.05); it was correlated negatively to the expression of IFN-γ in P-jejunum, ileum, and cecum (p < 0.05). Actinobacteria and Cyanobacteria were correlated negatively to the expression of β-conglycinin-specific IgG in M-jejunum (p < 0.05). Cyanobacteria was correlated negatively to the level of total serum IgG, IgE, and IL-10 in ileum (p < 0.05).We also performed an association analysis based on the Pearson’s rank correlation coefficient using different taxa by pooling these three groups together. At the phylum level Table , FirmicuLactobacillus was correlated negatively to total serum IgG, IgE, and IL-10 and was correlated positively to IFN-γin all intestinal segments. Clostridiaceae, Peptostreptococcaceae, and Enterobacteriaceae were correlated positively to the expression of β-conglycinin-specific IgG, IL-4, and IL-10 (p < 0.05) and were correlated negatively to the level of IFN-γ in M-jejunum (p < 0.05). Clostridiaceae and Peptostreptococcaceae were correlated positively to the level of IL-10 (p < 0.05) and were correlated negatively to IFN-γ in ileum. Enterobacteriaceae was correlated positively to β-conglycinin-specific IgG, glycinin-specific IgG, IL-4, and IL-10 (p < 0.05) and correlated negatively to IFN-γ (p < 0.05) in cecum. Streptococcus was correlated positively to glycinin and β-conglycinin-specific IgG and IL-10 in P-jejunum (p < 0.05). Total serum IgG and IgE were correlated positively to Peptostreptococcaceae, Turicibacter, Streptococcus, Clostridiales, and Clostridium in all intestinal segments.At the genus level Table , LactobaSoybean antigen protein (particularly glycinin and β-conglycinin) often impairs the performance and immune function of swine , 2009, sPrevious studies have shown that soybean antigen-induced anaphylaxis mainly includes IgE-mediated type I allergic reaction and IgG-mediated type IV allergic reaction . GlyciniIn addition, total serum IgG and IgE antibody levels were increased in the allergy group, which were similar to the findings from other studies . PreviouThe analysis of serum cytokines showed that piglets with FOS supplementation had higher interferon-γ level, but lower levels of IL-4 and IL-10 compared with the allergy group. The IFN-γ is a symbolic cytokine of Th1 cells. Inflammation factors of IL-4 and IL-10 are secreted by Th2 cells, which stimulate the proliferation of B lymphocytes and generate IgE and IgG antibodies , which pFirmicutes and the genus of Lactobacillus were decreased in the allergy group. A previous study found a similar result that soybean antigen, such as β-conglycinin, can inhibit the proliferation of Lactobacillus and Bifidobacteria (Lactobacillus protects the host against potential pathogen invasions (Lactobacillus and Bifidobacteria (Lactobacilli and Bifidobacteria have been shown to improve the increased gut permeability during the exposure to food allergens (Lactobacillus was higher in M-jejunum and ileum, whereas Bifidobacteria also increased in M-jejunum. A recent research has shown that a chronic enrichment of Proteobacteria in the gut, including Enterobacteriaceae, can represent an imbalanced/unstable microbial community structure or a state of disease of the host (Proteobacteria are a major phylum of gram-negative bacteria, which can increase lipopolysaccharide endotoxin in the blood, decrease the number of intestinal barrier cells, and increase intestinal permeability, leading to chronic inflammatory response (Proteobacteria was significantly higher in the allergy group in all intestinal tracts and decreased in the FOS group at P-jejunum and ileum. Therefore, consumption of FOS might modulate the intestinal microbes, improve the gut permeability, and decrease the allergy reaction. We also observed Turicibacter enrichment in the FOS group at P-jejunum and ileum. Similarly, supplementation of stachyose and barley malt can increase the relative abundance of Turicibacter (Streptococcus, can increase the levels of IL-6, IL-8, and IL-10 (Streptococcus was increased significantly in the allergy group and decreased in the FOS group at P-jejunum and ileum.Gut microbes play a crucial role in the establishment of tolerance to food proteins. In this study, the microorganisms in the sensitized group changed significantly, indicating the damages to the intestinal microenvironment in piglets sensitized by soybeans. The phyla of bacteria . Lactobanvasions , promotenvasions , modulatnvasions . It was bacteria . Both Lallergens . In the the host . Proteobresponse . In our cibacter . Some pand IL-10 . Our stuLactobacillus was correlated negatively to the expression of IL-10 and total serum IgE and was correlated positively to the expression of IFN-γ. Previous studies reported that Lactobacillus suppressed T-cell proliferation and Th2 cytokines secretion from allergic objects (Proteobacteria promotes colonic inflammation (Proteobacteria and genus of Enterobacteriaceae were correlated positively to IL-4 and IL-10 and were correlated negatively to IFN-γ. Peptostreptococcaceae, Turicibacter, Streptococcus, Clostridiales, and Clostridium were correlated positively to total serum IgG and IgE.Studies show that microbes have modulatory effects on regulatory T-cells, which provides mutual benefits and regulations between the host immune system and microbes . We foun objects , inhibit objects , and enh objects . Proteobammation  and induammation . We founLactobacillus and Bifidobacteria in M-jejunum and the decrease in Proteobacteria in P-jejunum and ileum. In addition, FOS can increase the total serum IgG and IgE to enhance the immune function. Our results provide a new and effective solution for the alleviation of allergic symptoms in livestock and humans and provide new hope for the development of prebiotics for the prevention and treatment of allergic reactions.Our study demonstrates that oral supplementation of FOS can effectively relieve anaphylaxis induced by the soybean antigen in piglets. This protective mechanism is associated with the suppression of specific IgG and inflammatory cytokine release as well as the change in gut microbes. Improvement of intestinal microbes is mainly indicated by the increase of MC and YZ designed and wrote this manuscript. XZ was involved in modifying the manuscript. GQ supervised the writing of this manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose: Hydroxyurea (HU) is a well-known chemotherapy drug with several side effects which limit its clinical application. This study was conducted to improve its therapeutic efficiency against breast cancer using liposomes as FDA-approved drug carriers.Methods: PEGylated nanoliposomes-containing HU (NL-HU) were made via a thin-film hydration method, and assessed in terms of zeta potential, size, morphology, release, stability, cellular uptake, and cytotoxicity. The particle size and zeta potential of NL-HU were specified by zeta-sizer. The drug release from liposomes was assessed by dialysis diffusion method. Cellular uptake was evaluated by flow cytometry. The cytotoxicity was designated by methyl thiazolyl diphenyl-tetrazolium bromide (MTT) test.Results: The size and zeta value of NL-HU were gotten as 85 nm and -27 mV, respectively. NL-HU were spherical.NL-HU vesicles were detected to be stable for two months. The slow drug release and Weibull kinetic model were obtained. Liposomes considerably enhanced the uptake of HU into BT-474 human breast cancer cells. The cytotoxicity of NL-HU on BT-474 cells was found to be significantly more than that of free HU.Conclusion: The results confirmed these PEGylated nanoliposomes containing drug are potentially suitable against in vitro model of breast cancer. Where nanosize liposomes would pass the tumors vasculature and accumulate in large quantities.-16 Several efforts have been made in this field. In a study, Alavi et al prepared and evaluated liposomal hydroxyurea on MCF7 cells.17 O’Shaughnessy et al also showed the successful use of PEGylated liposomes containing doxorubicin for breast cancer treatment.18 In the present investigation, HU-loaded PEGylated nanoliposomes were prepared and, first, assessed on BT-474 breast cancer cell.Breast cancer is one of the most prevalent diseases, with a high death rate among women.Dipalmitoyl phosphatidylcholine (DPPC) and methoxy-polyethylene glycol-derivatized di- stearoyl phosphatidylethanolamine (DSPE-mPEG2000) were obtained from Lipoid GmbH . Fluorescein isothiocyanate (FITC), HU and cholesterol (Chol) were provided from Sigma-Aldrich , Sucrose, Chloroform, and methanol were prepared from Merck . Polycarbonate screens were supplied from Northern Lipids . Streptomycin-penicillin-glutamine (S/P/G), SephadexG-50 were taken from Invitrogen . All other chemicals were of analytical grade. As well, the BT-474 cell line was bought from the American Type Culture Collection.Liposomes were manufactured via the thin-film hydration technique. Briefly, DPPC: Chol: DSPE-mPEG2000 at 7:4:0.18 molar proportions solubilized in a chloroform-methanol mix at a ratio (2:1).After evaporation of the solvents, the film was dried and suspended in phosphate buffer saline  containing HU (molar ratio drug/lipid = 0.2/1), stirred and warmed up to 50℃ in the water bath. The liposomes were mixed using a homogenizer  for 4 min at 16 000 rpm and extruded through polycarbonate screens 100 nm for ten circles by an Extruder . The NL-HU were segregated from free HU on a Sephadex G50 column. To determine the encapsulation efficiency (EE), one milliliter of purified NL-HU was disrupted by isopropanol (1:1 ratio) to release the drug.20:The value of drug encapsulated in liposomes was computed by a spectrophotometer  at 214 nm via plot standard curve and the following formula21 was executed to measure the lipid content in the nanoliposomal formulations. Lipids were solubilized in chloroform, followed by removing the solvent by a rotary motion. The thin film was hydrated in DW, 1 mL chloroform added and stored overnight in an oven at 90°C. Then was dissolved by 70 mL chloroform and immediately after centrifugation of the dispersals, we calculated the optical density (OD) of the standard and tests at 485 nm and detected the concentration by the standard curve.The Stewart assay,The size, zeta index, and polydispersity value of the NL-HU were examined by a zeta-sizer instrument  at 25°C in triplicate.Morphology of NL-HU was assessed by a scanning electron microscopy  and the transmission electron microscopy . For SEM analysis, a small quantity of liposomal suspension was scattered on a stub and parched at environment temperature. NL-HU were layered with gold by sputter coater  that is equipped with a rotary pump to attain vacuum less than 50 militorr, the proper vacuum range for noble metals, and sample coating with an additional thin layer (~10 nm) of the gold conductive material to gather high-quality information from SEM. Finally, were examined by SEM at 26 kV. For TEM study, one droplet of well-dispersed formulations was settled on the carbon-layered copper grid, wiped at environment temperature, and photographed using TEM at 150 kV.To specify the percentage of drug release, 1 mL of NL-HU and free HU were put into the dialysis bags (cut off: 14 000), immersed in 50 mL PBS (pH 7.4) and left on a stirrer for 36 hours at 37℃. At specific time intervals, 1 mL of reservoir buffer was taken and superseded by the same content of the fresh buffer. The amounts of HU released in the PBS were determined by spectrophotometer at 214 nm and the standard curve. The below formula computed the percentage of drug released (Er):HU illustrates the content of HU in the liposomes, V0 declares the whole volume of the release media and Ci and Cn manifest the concentration of HU in ith and nth aliquot removed, respectively.M2), release rate constants (kx), diffusion exponent (n), shape factor (b) were calculated. Well-fit kinetic equation with result data was considered as the best model.To designate the release mechanism from kinetic models including first order, zero order, Weibull, Higuchi, and Korsmeyer Peppas, the release data were analyzed using Excel 2017  Add-In DD Solver program. The coefficient of determination  were placed into six-well plates and incubated with the labeled free HU and NL-HU in the incubator at 37°C for 4 hours. The untreated cells were as the control. Finally, the cells were rinsed with cold PBS and appraised using the flow cytometry . WinMDI software was used to analyze the results.Internalization of the NL-HU and free HU into BT-474 cells was investigated by flow cytometry. Free HU and NL-HU were labeled by FITC. Briefly, 130 μL of PBS (pH = 7.4) and 65 μL of FTIC (2 mg in 1 cc DMSO) were poured into the free HU solution and NL-HU suspension and mixed gently. Unbounded FITC was separated by Sephadex G-50 column. Cells (300 × 1050 (50% inhibitory concentration of cell growth in comparison to untreated control cells as negative control),23 were determined. All tests were done in triplicate.The cytotoxicity of NL-HU against BT-474 cells was designated by MTT assay. Briefly, the cells were kept in RPMI medium containing S/P/G and 10% FBS in a 5% CO2 incubator at 37°C. Then, they were sub-cultured in 96-well plate (50 000 cells/well) in 100 µL of media at 37°C. After 24 hours of incubation, the cells were pasted to the wells. The free HU (as positive control) and NL-HU at the drug concentrations of 200, 400 and 600 µM were poured in the wells and incubated for 48 hours. Then the PBS-containing MTT (5 mg/mL) was added to the treatments and incubated for 3 hours; afterward, the formazan crystals were dissolved in 100 µL of DMSO. The OD was monitored using ELISA reader at 570 nm. Cell viability percentage and ICt test and one-way ANOVA followed by Tukey’s HSD post hoc test. Statistical significance was set at P< 0.05.Data were shown as mean ± standard deviation from three tests. The results were analyzed in SPSS software  with the student 17 The polydispersity index was equal to 0.12±0.030 for nanoliposomal HU; also, the mean zeta potential of the NL-HU was equal to -27 ± 0.51 mV (17 Lipid content of liposomes was equal to 89 ± 3.6%. Particle size and polydispersity index are fundamental factors for the preparation of proper nano-drug delivery systems; they contribute to the toxicity and delivery ability of these systems. For this purpose, the right size should be between 10 and 200 nm, where, the value of 85 nm was in this range due to the suitable choice of constituents, their proportions, and preparation method.28The size of NL-HU was approximately 85 ± 2.2 nm, which was lower than the previous report .17 The p 0.51 mV . NL-HU w29 Zeta potential is a significant criterion in the stability of liposome formulations and in blocking their aggregation, as well.30 Zeta potential of nanoliposomes was negative because of the existence of carboxylic groups of lipids, drug–to–lipid ratio, pH, and type of lipids used in the liposomes. The obtained zeta potential was enough to keep the stability of liposomal formulations. Based on SEM and TEM images, the NL-HU were found to be spherical due to the appropriate method of liposome preparation. The observed dimensions of liposomes using SEM and TEM were slightly smaller than those of the zeta-sizer because of zeta-sizer displays the hydrodynamic diameter of liposomes, but SEM and TEM depict the dried form of the liposomes.31 The EE% of HU was high because of the suitable HU-loading method and drug to lipid ratio.32Accordingly, the PDI was calculated based on the square of the ratio of standard deviation over diameter; the PDI <0.2 in this study indicates the high homogeneity of the liposomes.17 Drug release from NL-HU was significantly less than free HU because drugs are surrounded by liposomes that prevent them from being released. The liposomes exhibited fast drug release during the first ten hours. The reason for that is HU adsorbed on the liposome or the release of the drug-loaded close to the liposome surface. It was followed by a slow release due to liposome components, the liposome erosion, and HU diffusion mechanisms.33 The sustained release pattern from liposomes could diminish prescription times. The mathematical modeling of the release data for NL-HU was done as well, and the kinetic parameters listed in 37The pattern of drug release for free HU and NL-HU at pH = 7.4 was presented in As is seen in 38These findings were related to the suitability of the applied method, components, and their ratios of liposomes.42The results of quantitative cellular internalization analysis by flow cytometry in 45As is viewed in We designed and evaluated HU-loaded PEGylated nanoliposomes on an in vitro model of breast cancer. The results demonstrated this liposomal drug system could be potentially useful for delivery of the hydrophilic anticancer drug. Nevertheless, further investigations are needed to assess this formulation on other breast cancer cells, and in vivo.Not applicable.Authors declare no conflict of interest in this study.


Several studies examined the impact of miR-34b/c rs4938723 polymorphism and cancer risk, but the findings are inconsistent. However, no study has been conducted to inspect the impact of miR-34b/c polymorphism on bladder cancer. This study aimed to assess possible association between rs4938723 polymorphism and bladder cancer risk. This case-control study was done on 136 pathologically proven bladder cancer patients and 144 controls. Genotyping of Pri-miR-34b/c rs4938723 polymorphism was achieved by using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Our findings did not show any statistically significant differences in genotype and allele frequencies between bladder cancer and controls. Larger sample sizes with diverse ethnicities are required to validate our findings. Men are more than four times more likely to get bladder cancer than women. Bladder cancer has a multifactorial etiology. It has been proposed that the development of bladder cancer is a result of environmental factors such as smoking, occupational exposure to carcinogens, obesity, physical inactivity–4, genetic variants and the interaction of genes with the external factors–8.Bladder cancer is the ninth most common malignancy in the world, and the fourth most common cancer in the United States–10. The data provides strong evidence that dysregulation of miRNAs expression affects the tumorigenesis by acting as oncogenes or tumor suppressors–15. Single-nucleotide polymorphisms (SNPs) in miRNAs can affect cancer susceptibility by disturbing miRNAs biosynthesis and expression, altering mature miRNAs, or by combining with target genes–19.MicroRNAs (miRNAs) are a class of small single-stranded noncoding RNA molecules that play key roles in a variety of cellular processes by targeting mRNAs for cleavage or translational repression. A potentially functional rs4938723 variant (T to C substitution), located in the promoter region of pri-miR-34b/c, may affect miR-34b/c expression via genetic and epigenetic mechanisms and in turn influence the individual susceptibility to cancer–23. Though the association between miR-34b/c rs4938723 polymorphism and the risk of developing several cancers were reported in various case-control studies,23–34, but to the best of our knowledge, there is no report concerning the impact of miR-34b/c rs4938723 variant on the risk of bladder cancer. Accordingly, this case-control study was aimed to evaluate the possible association between pri-miR-34b/c rs4938723 polymorphism and susceptibility to bladder cancer in a sample from the Iranian population.The miR-34 family members comprises miR-34a, miR-34b, and miR-34c that are encoded by two different primary miRNAs. The miR-34a is encoded by its own transcript, while the miR-34b and miR-34c are encoded by a shared primary transcript (pri-miR-34b/c). All participants were from the Department of Urology, Shahid Labbafinejad Medical Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. The local ethics committee of Zahedan University of Medical sciences approved the project and informed us that written consent was obtained from all of the study participants. The genomic DNA was extracted from peripheral blood cells using the salting-out method.A total of 136 patients with histopathologically confirmed papillary urothelial cancers of the bladder and 144 healthy controls were enrolled in this case-control study. The study design and recruitment procedures were described previously. Briefly, the forward and reverse primers to amplify the Pri-miR-34b/c gene sequences containing the rs4938723 polymorphisms were 5′-CCTCTGGGAACCTTCTTTGACCTGT-3′ and 5′-CCTGGGCCTTCTAGTCAA-ATAGTGA-3′, respectively. PCR amplification was done using Prime Taq Premix  by the following amplification procedure: denaturation at 95 °C for 5 minutes, followed by 30 cycles of 95 °C for 30 seconds, 57 °C for 30 seconds, 72 °C for 30 seconds, with a final extension of 10 minutes at 72 °C. Ten microliters of the PCR products of 212 bp fragments were digested by NmuCI restriction enzyme (Fermentas) and separated by 2.5% agarose gel electrophoresis. The T allele was undigested (212 bp fragment), but the C allele digested and produced two fragments of 186 and 26 bp Fig. 1).( Genotyping of the Pri-miR-34b/c rs4938723 polymorphism was done by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique as explained previously2 and t-test, respectively. Odds ratio (OR) and 95% confidence interval (CI) was computed by unconditional logistic regression analysis to determine the association between the variant and the bladder cancer. The statistical level of significance was set as the P<0.05 level.Statistical analysis was carried out using statistical package SPSS 22 software. The categorical and continuous data were analyzed using χt>C polymorphism was successfully genotyped for 136 bladder cancer patients  with an average age of (63.8±12.3) years and 144 controls  with mean age of (64.3±10.2) years. No significant difference was found between the groups regarding age ( P = 0.701) and sex ( P= 0.076). The genotype and allele frequencies of pri-miR-34b/c rs4938723 polymorphism in bladder cancer patients and controls are shown in Table 1. The results indicated that pri-miR-34b/c rs4938723 t>C polymorphism was not associated with the risk of bladder cancer in codominant , dominant , recessive , overdominant  and allelic  inheritance model tested. We also calculated adjusted OR and 95%CI for sex and age Table 1) than that of patients with ages≤60 years (26.4%).The association between pri-miR-34b/c rs4938723 polymorphism and clinicopathological characteristics of bladder cancer patients are shown in 2 = 2.483, P = 0.115 and χ2 = 0.019, P = 0.88, respectively).The genotype rs4938723 polymorphism of pri-miR-34b/c in controls and cases were in HWE  and retinoblastoma. The variant has been shown to be associated with increased risk of papillary thyroid carcinoma (PTC) and nasopharyngeal carcinoma. The rs4938723 variant was found to be associated with increased risk of hepatocellular carcinoma (HCC) in the Chinese,40–41 and Korean populations.In the present study, for the first time, we inspected whether the pri-miR-34b/c rs4938723 et al. and Yang et al.  have found that pri-miR-34b/c rs4938723 variants significantly decreased the risk of gastric cancer (GC) in Chinese population,43. On the other hand, the findings of Wu et al.  did not support an association between the variant and risk of GC in Chinese population. Zhang et al. findings revealed that rs4938723 variant significantly decreased the risk of esophageal squamous cell carcinoma (ESCC) in the Chinese population. While, Zhu et al. has found no significant association between the variant and risk of ESCC in the Chinese population.Pan et al. have found no significant association between rs4938723 variant and colorectal cancer (CRC) in Korean population, while Gao et al. reported that this variant decrease the risk of CRC in Chinese population–47. The rs4938723 variant have been shown to be associated with increased risk of cervical cancer, renal cell carcinoma and prostate cancer.Oh et al. and Hashemi et al. reported that the rs4938723 variant significantly decreases the risk of childhood acute lymphoblastic leukemia (ALL).Tong There is no clear reason for the inconsistent findings regarding the association between pri-miR-34b/c rs4938723 variant and cancer risk. Ethnic, genetic, and/or environmental factors as well as gene-diet interaction may interact in various modes to either increase or decrease the risk of various cancers in different regions.In summary, our findings did not support an association between rs4938723 polymorphism in the promoter region of pri-miR-34b/c and the risk of bladder cancer in a sample from the Iranian population. Further large-scale studies with diverse ethnicities are warranted to reveal the impact of rs4938723 on bladder cancer.


ION) and low-level OFF-state current (IOFF); ambipolar current (IAMB). In detail, its ION is enhanced by 24 times more than that of Si control group and by 6 times more than of SiGe control group. The IAMB can be reduced by up to 900 times compared with the SiGe control group. In addition, technology computer-aided design (TCAD) simulation is performed to optimize electrical performance. Then, the benchmarking of ON/OFF current is also discussed with other research group’s results.In this paper, a novel tunnel field-effect transistor (TFET) has been demonstrated. The proposed TFET features a SiGe channel, a fin structure and an elevated drain to improve its electrical performance. As a result, it shows high-level ON-state current ( VDD) to be decreased drastically, maintaining high ON-state current (ION). In addition, its fabrication process is highly compatible with that of MOSFETs. In spite of these advantages, however, the TFETs have some technical issues to be employed for a real application. First, it suffers from low-level ON-state current which is mainly attributed to the high tunnel resistance at source-to-channel junction [IOFF); ambipolar current (IAMB). Since these issues degrade TFET circuit’s electrical performance such as operation speed and power consumption, they should be addressed [Numerous studies about tunnel field-effect transistor (TFET) have been performed by several research groups as a promising device for an ultra-low power operation ,2,3,4. Ijunction ,10,11. Ijunction . Howeverjunction . Second,ddressed ,14,15,16ION and smaller IAMB than that of conventional Si TFETs. As shown in ION as reducing tunnel resistance. In addition, in the proposed TFET, IAMB can be suppressed with the help of relatively large Si band gap at drain. In addition, its feasibility for better performance is examined by technology computer-aided design (TCAD) simulation. Last of all, based on the measurement and optimized results, the benchmarking of ON/OFF current ratio (ION/IOFF) and SS with the state-of-the-art TFETs is also discussed.The purpose of this paper is to demonstrate a novel TFET which achieves larger 2 wet etching. Then, SiGe and Si layers are grown on the SOI substrate by metal organic chemical vapor deposition (MOCVD). The process condition is as follow: a gas mixture of H2 at 20 sccm, SiH4 at 20 sccm, and GeH4 at 90 sccm is used at 670 °C during 61 s for 300 Å-thick SiGe. Auger electron spectroscope (AES) and transmission electron microscope (TEM) image confirm a single crystalline Si0.7Ge0.3 is well grown on Si substrate  as an etching mask during an active patterning (c). PECVD nitride is adopted since it is low temperature process with 400 °C and 20 s, in which the implanted dopants in the drain region can rarely diffuse. (d) Some part of Si on active regions are removed by photolithography and reactive ion etching (RIE) processes forming SiGe source and channel while the remaining Si on mesa becomes raised drain region. In case of channel, an additional patterning is conducted by mix-and-match process of e-beam lithography and photolithography to form 50 nm-width active fin (The key process steps for the proposed TFETs are described in ubstrate  As ion itive fin .4OH), hydrogen peroxide (H2O2), and de-ionized wafer (H2O) [4OH:H2O2:H2O ratio is 1:8:64 in which the etching rate of the SiGe is ~0.85 nm/min. After 13 min process in the SC-1 solution, the SiGe fin width is reduced to 39.5 nm as shown in the inset of 2 and SiGe [2 implantation with 10 keV-acceleration energy, 7°-tilted angle and 8 × 1014 ions/cm2-dose is performed for a source region. The dopant activation is performed by rapid thermal process (RTP) with 900 °C and 5 s. Note that all processes for gate, source and drain formation are self-aligned to each other and can be compatible with state-of-the-art ultra-short channel technology. Finally, as a back-end-of line (BEOL), high plasma density (HDP) oxide is deposited as an interlayer dielectric (ILD) and metal layers (Ti/TiN/Al/TiN stacks) are deposited by physical vapor deposition (PVD) after contact formation. (g) Then, all of processes are summarized in the flow graph.The SiGe/Si fin width is further reduced by standard cleaning-1 (SC-1) solution which consists of ammonium hydroxide (NHer (H2O) ,18. The and SiGe . The cap0.7Ge0.3 layers with a thickness of 30 nm are grown on SOI (100) substrates. The SOI layer is lightly p-doped (1 × 1015 cm−3) with a thickness of 70 nm. For additional comparison, the Si TFET is fabricated on a 100 nm-thick SOI wafer. The gate stack consists of 200 nm poly-Si layer and 3 nm SiO2. After gate patterning, source and drain region are defined through photolithography and ion implantation processes. The ion implantation and BEOL processes are same with the processes in proposed TFET.For the control samples, the planar Si and SiGe TFETs were fabricated. In the case of SiGe TFET, SiVDSs). The SS is extracted at VDS of 0.1 V and a turn-ON voltage (Vturn-ON) is defined as gate voltages (VGS) where BTBT first occurs. The IOFF and ION are extracted when VGS is Vturn-ON and gate overdrive (VOV = VGS − Vturn-ON) is equal to 2 V, respectively. The minimum SS is 81 mV/dec and ION/IOFF is 2.8 × 104. VGSs. Note that, the conventional planar devices suffer from short channel effect (SCE) due to their weak gate controllability over the channel [ channel ,21. GeneVDS. The SS and ION, IAMB and ION/IOFF are extracted from the curves and summarized in Vturn-ON is 81 mV/dec whereas 151 mV/dec and 87 mV/dec are measured in planar Si and SiGe TFETs, respectively. Second, the proposed TFET shows 139 nA/μm-ION which is 34 times and 5 times bigger than that for Si and SiGe TFETs, respectively. Last of all, the IAMB can be reduced by up to 103 times compared with the SiGe TFET. These results are attributed in part to the SiGe’s narrow bandgap at the source area and in part to the strong gate-to-channel coupling with the help of fin-structured channel [The proposed TFET’s electrical characteristics are compared with that for planar Si and SiGe TFETs as control groups.  channel . In addi channel .IOFF of proposed TFET near zero VGS is higher than that of planar Si TFET (ID) is relatively independent to the VGS at around 0 V while it increases rapidly as a function of temperature. The result confirms that this current is dominated by Shockley–Read–Hall (SRH) generation–recombination [The proposed TFET has remarkable electrical characteristics as shown above. However, the  Si TFET . In ordebination .ION and low IAMB. Compared with planar TFETs which is fabricated with the same processes, there is no doubt that the proposed structure is effective to improve electrical performance. However, the measured results imply that it requires further optimization for the better performance than the other strategies [G) per unit volume in the uniform electric field, Kane’s model is used and fitted parameters are as follows (Equation (1)).F0 = 1 V/m, P = 2.5 for indirect BTBT. Prefactor A and exponential factor B are the Kane parameters and F is the electric field. Both linear and log scale simulated transfer characteristics are well matched to experimental data when A: 1 × 1014 cm−1·s−1/B: 3 × 106 V/cm are applied to TFETs. Then, the thickness of the gate dielectric is analyzed. Unlike advanced technologies, the proposed TFET uses 3.4 nm thick SiO2 as the gate dielectric. Thus, if the gate dielectric is adjusted to 1 nm, the proposed TFET can obtain higher ION at the low VGS (ION/IOFF as a function of SS for the device shown in this paper and that in the previous articles [ION/IOFF.The objective of this study is to demonstrate the TFET with high rategies ,35,36,37rategies . In the  low VGS . Figure articles ,35,36,37ION and low SS, compared to conventional planar TFETs. Furthermore, the elevated drain could yield lower IAMB due to the increased physical distance between channel and drain. Considering these features, we have examined and demonstrated the fabrication processes of the proposed device. In addition, based on the measured results, the proposed TFET is calibrated by TCAD simulation. In order to optimize the device into state-of-the-art technique, the proposed device with thin gate dielectric is also simulated. The results proved that the device showed the improved ION current and smaller SS. Consequently, these features of the proposed device will be available for compensating the weaknesses of conventional TFETs. Therefore, it will be one of the promising candidates for next-generation devices.In this paper, a novel TFET with SiGe fin channel and elevated drain has been introduced. The SiGe fin channel included small-bandgap and better electrostatic controllability which are leading high


Fusarium, Myrothecium, and Stachybotrys species, causes dystrophy in the brain, heart, and kidney. Various formulations that include lemongrass essential oil-modified montmorillonite clay (LGEO-MMT), lemongrass powder (LGP), montmorillonite clay washed with 1 mM NaCl (Na-MMT), montmorillonite clay (MMT), and lemongrass powder mixed with montmorillonite clay (LGP-MMT) were applied to maize at concentrations of 8% and 12% and stored for a period of one month at 30 °C. Unmodified montmorillonite clay and LGP served as the negative controls alongside untreated maize. Fourier Transform Infrared (FTIR) spectra of the various treatments showed the major functional groups as Si-O and -OH. All treatment formulations were effective in the decontamination of T-2 toxin in maize. Accordingly, it was revealed that the inclusion of Na-MMT in maize at a concentration of 8% was most effective in decontaminating T-2 toxin by 66% in maize followed by LGP-MMT at 12% inclusion level recording a 56% decontamination of T-2 toxin in maize (p = 0.05). Montmorillonite clay can be effectively modified with plant extracts for the decontamination of T-2 toxin.Montmorillonite clay has a wide range of applications, one of which includes the binding of mycotoxins in foods and feeds through adsorption. T-2 toxin, produced by some  Fusarium langsethiae and F. sporotrichioides are some of the Fusarium species that infect crops in the field and storage during which time they produce T-2 toxin [Fusarium sporotrichioides, F. graminearum, F. poae, and F. culmorum and causing diarrhea, inflammation, and emesis [Maize is grown worldwide, with the United States, China, and Brazil ranked as the leading producing countries contributing approximately 563 of the 717 million metric tonnes per year . Fusariud emesis ,5. They d emesis . The cheT-2 toxin is a recognized inhibitor of protein synthesis, which occurs by the binding of peptidyl transferase, present in the 60S ribosomal subunit . T-2 poiDecontamination processes involving physical, biological, and chemical methods are frequently required for the removal, destruction, and decontamination of mycotoxins . Biologi4) shares three out of four of its oxygen atoms with the central octahedral sheets [Montmorillonite clay has pharmaceutical applications where it serves as an excipient in drugs for oral or topical use . The cytl sheets . Among vl sheets ,26.1, and ZEA [Cymbopogon citratus (lemongrass) has been expended in the decontamination of Aspergillus species and limit AF production in maize [Cymbopogon citratus can be safely used in the decontamination of mycotoxins in cereals. The aim of this study, therefore, was to decontaminate T-2 toxin in maize using formulations from montmorillonite clay together with extracts and powder of Cymbopogon citratus.The use of plant extracts may have ancillary effects, thus preventing fungal growth and the production of mycotoxins. The oils of plants such as anise, basil, cinnamon, lemon, clove, lemongrass, spearmint, and oregano have been shown to inhibit fungi that produce DON, FB and ZEA . Cymbopoin maize . Traditiin maize  with no in maize  revealedCymbopogon citratus.The scanning electron micrographs of the various treatment formulations with a magnification of 500× and scale bar of 100 µm are shown in −1 and a little depression along 3392.10 cm−1 for all the treatments containing montmorillonite clay. The bands at 1737 cm−1, 1620 cm−1, and 1515 cm−1 in LGP, as shown in −1 corresponds to the O-H stretch, which is an aliphatic stretch in moieties of cellulose. The band at about 1739 cm−1 is due to the C=O stretching of the acetyl group and linked to the ester linkage of the carboxylic group of the ferulic acid and p-coumaric acid of lignin [−1 represents that of an aliphatic compound attached to a hydroxyl (OH) group [−1 [-1 represent the bands for Si-O-Si bending and stretching, respectively [−1 represent the stretching of the -OH (hydroxyl group) of water within MMT [The Infrared spectra of lemongrass essential oil-modified montmorillonite clay (LGEO-MMT), lemongrass powder (LGP), sodium montmorillonite (Na-MMT), montmorillonite clay (MMT), and lemongrass powder mixed with montmorillonite (LGP-MMT) are shown in f lignin . The broH) group . The OH roup [−1 . The infT-2 toxin had an apparent recovery of 116%. Its limit of detection (LOD) was 0.002 µg/kg, while its limit of quantification (LOQ) was 0.006 µg/kg. The various types of treatment  applied to the maize sample from the same batch were effective over the treatment period of four weeks. The storage temperature (30 °C) was maintained for the treated maize samples and the control (untreated maize) throughout the four weeks of storage. The experiments were run for four weeks to establish the trend in the decontamination of T-2 toxin with time. The control (untreated maize stored under similar conditions as treated samples) remained unchanged throughout the treatment period from the first to the fourth week with a concentration of 30 ppb .p = 0.05). Montmorillonite clay, which has high adsorptive properties along with the positive charge of sodium (Na+), could have led to the effective trapping of T-2 toxin. It appears that the decontamination of T-2 toxin is better achieved by adsorption. Regular van der Waals gap between the sheets of clay is referred to as the interlayer spaces that possess a net negative charge due to ionic substitutions in the sheets of clay minerals [The Post-Hoc analysis data generated showed significant differences among the five treatments against T-2 toxin. Further statistical analysis using LSD to compare mean values among various treatment formulations for the decontamination of T-2 toxin revealed that sodium montmorillonite (Na-MMT) was the most effective against T-2 toxin  was more effective than the unmodified montmorillonite (MMT) due to the presence of Na species . LemongrCymbopogon citratus in combination with montmorillonite clay contributed towards making montmorillonite clay more hydrophobic than the unmodified montmorillonite. Erminawati et al. [For LGP-MMT at 12%, which was the second most efficient in the decontamination of T-2 toxin, the powder of i et al.  revealedi et al. . Type A-i et al. . T-2, beRhodococcus erythropolis, Rhodococcus globerulus, Rhodococcus rhodochrous, and Rhodococcus coprophilus. These bacteria were 90% efficient in the degradation of T-2 toxin, although their degradation products were not studied [Pseudomonas, Arthrobacter, Blastobacter, and those from the family Rhizobiaceae, have also been employed in the elimination of T-2 toxin with 100% efficiency recorded. However, Wachowska et al. [Microbiological approaches for the decontamination of T-2 toxin include the use of bacteria such as  studied . Other ba et al.  reporteda et al. . Hence, a et al. .+) ion, which made the clay electrically neutral and able to trap T-2 toxin especially, with sodium montmorillonite (Na-MMT) noted as the most efficient in decontaminating T-2 toxin in maize. Hence, the trapping of T-2 toxin was achieved by the regular van der Waals forces between the sheets of Na-MMT, which were enhanced by the Si-O bonds for the entrapment of this toxin within its layers. Adsorption processes for the decontamination of T-2 appear more promising than the biological methods for its decontamination because there could be very limited chances for the formation of toxic products.Montmorillonite clay was successfully modified on the surface with a sodium , Formic acid , Hexane ; Acetone , Sodium chloride salt, and deionized water. T-2 mycotoxin standard was purchased from Sigma-Aldrich . Maize samples were purchased from one of the grain hubs in Ogun state, and a screening was performed to identify a maize sample that was naturally contaminated with T-2 toxin. The naturally contaminated T-2 maize sample was utilized for the experiment.Cymbopogon citratus (lemongrass) leaves used for the study were identified at the Botany Unit of the Department of Biological Sciences, Covenant University, Nigeria.Montmorillonite K10 powder (CAS number 1318-93-0 (Sigma-Aldrich) was purchased, while Cymbopogon citratus were gently washed with distilled water to remove dirt, and the leaves were allowed to air-dry at room temperature for 3 weeks. The dried leaves were ground into powder by using an electric blender . The lemongrass extracts were obtained by the Soxhlet extraction method described by Ojewumi et al. [−1), at a solid:liquid ratio of 1/20. After that, the mixture was dialyzed in deionized water to eliminate chloride ions. The dialyzed samples were gently dried at 40 °C to allow for a gradual loss of water from the clay. In a 500 mL flask, 25 g of the clay powder was dispersed in 50 mL of essential oil solution (v/v) prepared with acetone. The mixture was whirled at room temperature (25 °C) in an overhead shaker at 50 revolutions per min. Afterward, the mixture was placed in a water bath operated at 60 °C for 90 min to allow for the evaporation of acetone. The mixture (LGEO-MMT) was dried overnight at 30 °C, and then stored in colored and tightly sealed vials using aluminum foil.Montmorillonite clay K10 and lemongrass powder were the negative controls for the treatment. Fresh leaves of i et al. . The dryi et al.  with slii et al. . The monw/v), while LGP-MMT was prepared by mixing lemongrass powder with montmorillonite clay in the ratio 1:1.For the Na-MMT mixture, montmorillonite clay was modified by washing the clay with 1 mM NaCl solution in the ratio 1/20 (Fourier Transform Infrared (FTIR) Spectroscopy of the clay samples was performed with the aid of a Perkin Elmer Spectrum 100 FTIR Spectrometer. One drop of Nujol (a liquid hydrocarbon) was added to 1 mg of each sample and mixed thoroughly. The mull was then placed between sodium chloride plates, and the spectrum was recorded.The samples were coated with carbon and examined with the aid of VEGA3 TESCAN scanning electron microscope at a voltage of 20 kV and magnification of 500× to examine their morphology.For the method validation of T-2 toxin, the apparent recovery (AR) was obtained by spiking a known amount of T-2 toxin to a blank. The blank was spiked with 0.25 and 1 µL of the T-2 reference standard within a calibration range of 156.25–5000 µg/kg. The limit of detection (LOD) and limit of quantification (LOQ) were also determined.v/v/v) for 90 min on a GFL 3017 rotary shaker . Five hundred µL of each filtered extract was then injected into the LC-MS/MS system to determine the level of T-2 toxin.Liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol, as reported by Sulyok et al. , was useEach of the various treatment formulations  were applied to the maize sample in duplicates at 8% and 12% and stored for 4 weeks at a temperature of 30 °C in the incubator following the modified method of Atanda and Olopade . A contrp ≤ 0.05.The efficacy of the various types of treatment, which were applied in duplicates, was determined by Post-Hoc analysis using the IBM SPSS statistics software . Subsequently, mean values among the various treatment formulations for the decontamination of T-2 toxin were compared by the Least Significant Difference (LSD) test and considered to differ at